Herpes-group viruses have recently been discovered to encode a ubiquitin-specific cysteine protease, whose biological function is unknown. Its catalytic domain lies within a 300 to 400 amino-acid sequence that is the amino terminal end of the largest protein encoded by each herpesvirus genome. Although its activity is not absolutely essential for virus replication, it appears to enhance the efficiency of the process. Our discovery that this deubiquitinylating enzyme (DUB) is active in the context of the full-length protein led to two key findings on which the work proposed here is based. First, the DUB is an integral constituent of infectious virions and second, it is the predominant if not sole DUB activity present in purified virions. We are studying the herpesvirus DUB in human cytomegalovirus (HCMV, HHV5) because this sexually transmissible agent is of increasing medical relevance in association with AIDS, organ transplantation, cancer chemotherapy, and birth defects resulting from transplacental infections. It is also the prototypic 2-herpesvirus and there is an underlying need to identify and understand differences between it and members of the 2- and 3- herpesviruses. In human cytomegalovirus the protein hosting the DUB activity is encoded by open reading frame UL48, is 253 kDa, and is called the high-molecular-weight protein (HMWP) or pUL48. The specific aims we propose follow directly from results of published and pilot studies done to define the biological purpose of this enzyme and determine its potential as an antiviral target. Our immediate aims are to substantiate the hypothesis that HCMV virions contain a substrate of the viral DUB (critical to understanding function and mechanism, but so far unidentified), and to develop an assay that can be adapted to high-throughput format to screen for inhibitors of the HCMV DUB. We will apply a combination of genetic, physical, biochemical, enzymological, and biological approaches to accomplish these aims in a multidisciplinary environment. PUBLIC HEALTH RELEVANCE: Cytomegalovirus is a herpes-group virus that is a threat to people with weakened immune systems, including the very young and the very old. This research is directed at a recently discovered protease that removes ubiquitin from proteins, but whose function and potential as an antiviral target are unknown. We propose to identify the substrate(s) of this enzyme and identify inhibitors of its activity in high-throughput assays.